环境因子对眼斑拟石首鱼精子活力的影响

通过测定精子的激活率、运动时间及寿命,研究了pH、盐度、离子及葡萄糖等因子变化对眼斑拟石首鱼(Csiaenops ocellatus)精子活力的影响。结果表明,眼斑拟石首鱼精液中精子平均浓度为(1.203±0.22)×10¹⁰个·mL^-1;精子激活与运动的适宜盐度范围为20～35,其中盐度25时精子的激活率、运动时间及寿命分别达94.32%、9.14min及12.55min;精子激活与运动的适宜pH为6.0～8.5,适宜的KCl、NaCl及CaCl₂溶液浓度分别为600～700mmol·L^-1、600mmol·L^-1及400～600mmol·L^-1,适宜的葡萄糖溶液浓度为700～900mmol·L^-1;精子在缺少HCO₃^-或Mg²⁺或Ca²⁺的人工海水中激活率与在人工海水中的激活率无显著差异,但运动时间和寿命有所缩短。     

Na+、葡萄糖、果糖对凹目白鲑精子活力的影响

研究了凹目白鲑（Coregonus autumnalis）精子在302．6～488．9kPa时的NaCl溶液、D-葡萄糖溶液及D-果糖溶液中的活动情况。结果显示,在NaCl溶液中凹目白鲑精子在395．7kPa具最佳活力,其快速运动时间（fast movement time,FT）与寿命时间（1ife time,LT）的最大值分别达43．57s和90．31s;与相同渗透压下NaCl溶液相比,凹目白鲑精子在葡萄糖和果糖溶液中的FT与LT均有不同程度的增加;在葡萄糖溶液中FT与LT的最大值分别为57．67s和97．29s,在果糖溶液中则为70．44s和105．29s。说明凹目白鲑精子活力不但与溶液渗透压有关,而且与溶液溶质有关。     

环境因子及超低温冻存对黄姑鱼精子活力的影响

通过测定精子的激活率、运动时间及寿命研究了环境因子变化对黄姑鱼精子活力的影响及超低温冻存后黄姑鱼精子的活力。结果表明,黄姑鱼精子激活与运动的适宜盐度为25～35、适宜pH为7.5～8.5。在pH 8.0～8.5、盐度25条件下,精子激活率达(85.33±2.52)%,运动时间及寿命分别为(336±14.02)s及(405.33±12.22)s。精子激活与运动的适宜NaCl、KCl、MgCl₂及葡萄糖溶液浓度分别为300～500 mmol·L^-1、600 mmol·L^-1、800～1000 mmol·L^-1及900mmol·L^-1;精子在缺少HCO₃^-的人工海水中未能被激活;精子在无Ca²⁺或无Mg²⁺的人工海水中激活率均大于80%,但运动时间及寿命均有所缩短。以Cortland及HBSS溶液为稀释液、10%EG为抗冻剂冻存黄姑鱼精子,冻精激活率>80%,运动时间均超过200s。     

钠、钾、钙和葡萄糖对白斑狗鱼精子活力的影响

观察了白斑狗鱼精子在0～679．6kPa NaCl、KCl、葡萄糖溶液和0～339．8kPa CaCl2溶液中的活动情况。在NaCl、KCl、葡萄糖溶液中,白斑狗鱼精子快速运动时间和寿命的变化规律基本一致,精子活动最适渗透压介于339．8～453．0kPa。K^ 有延长精子寿命的作用。外源性葡萄糖可被精子利用,增强精子活力．延长精子寿命。56．7kPa CaCl2对精子活动有抑制作用,并引起精子聚集,该效应随着Ca^2 浓度升高而增强。     

不同渗透压的稀释液对猕猴精子低温冷冻保存的影响

以稀释液TTE(382mOsm/kg)为对照,研究了5种渗透压(688、389、329、166、43mOsm/kg)的TEST稀释液(TEST、mTEST1、mTEST2、mTEST3、mTEST4)在冷冻过程中对猕猴精子功能的影响。精液一步稀释于含甘油的防冻液中,甘油的终浓度为5%(v/v)。在冷冻前后分别检测精子的运动度和质膜完整性,后者用Hoechst33342和碘化丙锭双色标记流式细胞术分析。结果表明:冷冻之前,与鲜精相比,用TEST和mTEST4稀释的精子运动度和质膜完整性显著降低(P<0·001),其余组中除mTEST2稀释的精子质膜完整性显著降低(P<0·05)外,精子运动度无差异;冷冻复苏后,TTE、mTEST3和mTEST1冻存精子的运动度和质膜完整性最高,其次是mTEST2,TEST和mTEST4冷冻效果最差(P<0·05)。提示等渗、适当高渗或低渗的稀释液适合猕猴精子的冷冻保存;对精子产生高渗毒害作用是导致猕猴精子用TEST冷冻存活率低的主要原因。     

黑玛丽精子包特性及pH和温度对其精子活力的影响

通过研究黑玛丽Poecilia latipinna精子包破裂的过程和影响因素、精子运动的情况及影响因素,结果发现,当精液用Hank’s平衡盐溶液(HBSS)稀释约5 min后精子包开始破裂,约12 min后全部破裂。释放出的精子暂时处于休眠状态,约50 min后,处于HBSS稀释液中的精子会被激活。应用计算机辅助精子分析系统对黑玛丽精子在不同pH和不同温度下HBSS中的精子运动百分数、运动时间和平均运动速率进行观察。在pH7~8的中性或弱碱性溶液中,精子运动活力较强;而在酸性(pH<7)或碱性较强(pH>9)的溶液中,精子的运动活力都会降低。在不同温度下的HBSS中精子的活力不同,精子在低温(4℃)条件下的运动时间显著长于在室温(20℃)条件下,但运动速度较慢。本研究初步探讨了黑玛丽的精子包特性以及pH和温度对精子运动活力的影响,旨在对黑玛丽等卵胎生鱼类的人工授精,以及生殖生物学特性等的研究提供更丰富的基础资料。     

中华蟾蜍蝌蚪对高渗溶液的耐受性

为了解中华蟾蜍蝌蚪对高渗溶液的耐受性,采用常规静态实验,用蔗糖调节溶液渗透压对蟾蜍蝌蚪进行渗透耐受实验;采用方差分析统计溶液渗透压对蟾蜍蝌蚪的存活率、体积变化和活动性的影响。结果显示,蟾蜍蝌蚪能够忍受溶液渗透压在一定范围内的变化,对渗透压较低的溶液其适应对策为调节,而对渗透压较高的溶液则为协变。蝌蚪对水溶液的耐受渗透压为1485．24mOsm,其存活率随渗透压增高而降低;同一渗透压下,蝌蚪存活率随在溶液中暴露时间的延长而降低。蟾蜍蝌蚪体内水分在不同浓度溶液中丧失的程度存在显著差异,即使在同一浓度的溶液中,体积会随时间的增加而不断缩小,并且溶液浓度越高,体积减小的速率越大。身体失水对蝌蚪的行为性会产生一定的影响,蝌蚪的活泼性随时间的延长而降低。     

不同发育时期中华绒螯蟹血淋巴渗透压的分析

中华绒螯蟹血淋巴渗透压随不同的生长阶段而异;不论是在淡水中,还是在不同盐浓度的海水中,都具有保持血淋巴高渗调节控制的能力。青春期前,6—9月份,血淋巴渗透压自390mOsm/Kg H_2O升至560mOsm/Kg H_2O;青春期后,9—12月份,自555mOsm/Kg H_2O升至656mOsm/Kg H_2O,但12月到翌年3月份却明显下降。雌蟹从淡水移入海水后的血淋巴渗透压调升速度,青春期后是青春期前的三倍左右。     

TRPV4在渗透压对大鼠心肌收缩性影响中的作用

本文旨在探讨低渗和高渗内环境对心肌收缩性的影响及机制.取Sprague-Dawley(SD)大鼠左心室乳头状肌,在电刺激引起兴奋的条件下,分别记录在低渗、等渗和高渗灌流液中肌条的收缩力;同样条件下观察在低渗、等渗和高渗灌流液中加入渗透压敏感蛋白瞬时感受器电位离子通道家族香草素受体亚家族IV型(transient receptor potential vanilloid 4,TRPV4)的拮抗剂和激动剂后肌条收缩力的变化.结果显示:(1)与等渗(310 mOsm/L)时心肌收缩力相比,渗透压为290、270和230 mOsm/L时心肌收缩力分别增加11.5%、21.5%、25.O%(P<0.05);渗透压为350、370、390 mOsm/L时心肌收缩力分别降低16.0%、23.7%、55.2%(P<0.05).(2)在低渗液(270 mOsm/L)中加入TRPV4拮抗剂钌红(ruthenium red,RR),低渗对心肌收缩力的增强作用被抑制36%(P<0.01);在高渗液(390 mOsm/L)中加入RR,高渗对心肌收缩力的抑制作用增加56.1%(P<0.01).(3)在等渗液中(310 mOsm/L)加入TRPV4激动剂4-α-佛波醇-12,13-二癸酸(4-α-phorbol-12,13-didecanoate,4 α-PDD),心肌收缩力没有改变;在高渗液中(390 mOsm/L)加入4α-PDD,高渗对心肌收缩力的抑制作用增加27.1%(P<0.01).以上结果提示,TRPV4参与渗透压引起的心肌收缩力变化.     

青岛文昌鱼精子运动特征及计算机辅助分析

青岛文昌鱼的精子在精浆中不运动,在与精浆等渗的生理溶液中也不运动,把精液稀释到过滤海水或人工海水后,精子运动被激活。文昌鱼新鲜精液中精子浓度为(9.4±1.6)×1010/ml;在人工海水中,运动精子占总精子数的85.8%±6.9%,精子运动持续的平均时间是22.7±2.6min。文昌鱼精子经人工海水激活0.5min后,可观察到四种运动方式:(1)向前直线运动,其平均速度93.6±23.7μm/s,这类精子所占的百分比为27.8%±3.1%;(2)弧形曲线运动,平均速度55.6±18.9μm/s,所占的百分比为44.3%±2.5%;(3)左右摆动,平均前进速度27.4±13.4μm/s,所占百分比为14.7%±1.8%;(4)运动速度小于5μm/s的精子,视为不运动的精子,所占百分比为13.2%±1.8%。随着激活时间的延长,文昌鱼精子的运动状态发生改变,向前直线运动和弧形曲线运动的精子逐渐减少,而左右摆动和不运动的精子逐渐增多。     

Changes in extracellular osmolality initiate sperm motility in freshwater teleost rosy barb Puntius conchonius

The objective was to investigate the effects of extracellular osmolality and membrane osmotic-sensitive channels on the initiation of sperm motility and to explore mechanisms of sperm initiation in rosy barb (Puntius conchonius). We found that (1) sperm were immotile in seminal plasma and remained quiescent in electrolyte or nonelectrolyte solutions isotonic to seminal plasma; (2) sperm movement was initiated when the sperm were exposed to hypo-osmotic electrolyte or hypo-osmotic nonelectrolyte solutions, and that the responsiveness of sperm to changes in the extracellular osmolalities (100, 200, 250, 270, and 300 mOsm/kg) differed among sperm cells (P < 0.05); (3) sperm movement could be initiated and terminated repeatedly by decreasing and increasing the osmolality (in increments of 100 and 300 mOsm/kg) of a nonelectrolyte mannitol solution, respectively (P < 0.05); (4) gadolinium (20, 40, and 80 μM) inhibited the initiation of sperm motility and abolished the sperm activation caused by the hypo-osmotic media treatment in dose- and time-dependent manners (P < 0.05); and (5) sperm activation in a hypo-osmotic medium and inhibition in an isotonic solution were associated with swelling and shrinkage of the sperm sleeves, respectively. Therefore, we concluded that osmolality was a critical physiologic signal in regulating the initiation and termination of sperm motility in freshwater teleost rosy barb. Furthermore, we inferred that rosy barb sperm were hypo-osmotic–dependent conformers, and the osmotic-sensitive channel could be involved in the mechanism of sperm initiation.     

Use of heterologous sperm for the dispermic induction of androgenesis in barbs

Dispermic activation of genome‐inactivated eggs of the grey tiger barb Puntius tetrazona , facilitated by 2·5% polyethylene glycol (PEG)‐incubated sperm of the golden rosy barb Puntius conchonius , resulted in the generation of interspecific androgenetic clones of the golden rosy barb. A 10 min incubation of the golden rosy barb sperm in increasing concentrations (1·0, 1·5, 2·0, 2·5 and 3·0%) reduced the frequency of motile sperm (to 70%), motility duration (110–50 s) and fertilizability (to 80%) of the sperm; however, the frequency of sperm with double head size increased. At 3% PEG, motility pattern of the sperm completely changed from 'zig‐zag' to 'circular'. Incubation in 2·5% PEG facilitated the dispermic entry and production of diploid androgenetic female and male progenies at the ratio of 0·27 : 0·73. Polymerase chain reaction (PCR) analysis using Tc1 transposon specific primers confirmed the purity of paternal inheritance of P. conchonius through the surrogate eggs of P. tetrazona . Survival and breeding, body colour, diploidy (karyotyping and erythrocyte measurements) and 0·27F : 0·73M sex ratio of the androgenotes provided evidence for the successful induction of dispermic androgenesis. Despite increased heterozygosity and reduced 'damage cost' on restoration of diploidy, survival of dispermic androgenotes induced in heterologous eggs was lower (1·7%) than those reported for androgenetic golden rosy barbs induced using homologous sperm (14·0%) and heterologous eggs (7·0%), i.e . tiger barb eggs.     

Ions and osmolality on post‐thaw sperm motility activation of the endangered Brycon insignis (Characiformes)

The aim of this study was to evaluate the effects of osmolality and the presence of ions on the activation of post‐thaw sperm motility of Brycon insignis. Sperm was frozen under a standardized methodology for this species. In experiment 1, 11 solutions were prepared with reverse osmosis (RO) water (～0 mOsm/kg) and glucose or NaCl adjusted to an osmolality of 50, 100, 150, 200 and 250 mOsm/kg. In experiment 2, six solutions were prepared with RO and adjusted to ~100 mOsm/kg with one of the following chemicals: NaHCO₃, sodium citrate (Na₃C₆H₅O₇), NaCl, KCl, CaCl₂ or glucose (as ion‐free control). Post‐thaw sperm of both experiments was evaluated for motility rate, velocities (curvilinear = VCL, among others) and beat‐cross frequency (BCF). In experiment 1, sperm motility rate and velocities were higher (p < 0.05) when triggered in solutions at osmolalities from 0 to 200 mOsm/kg (62–80% motility; 139–167 µm/s) than that at 250 mOsm/kg (36–44% motility; 94–99 µm/s VCL). BCF was not affected by osmolality and varied from 19 to 24 Hz in all samples. In experiment 2, samples activated in NaHCO₃, citrate, NaCl and KCl solutions yielded higher motility rates (76–85%) and BCF (24–25 Hz) compared to those activated in CaCl₂ (50%; 14 Hz). Samples activated in ion‐free control solution yielded higher motility rate (87%) than those activated in NaHCO₃ and in CaCl₂. Curvilinear velocity was higher in samples activated in NaHCO₃, citrate, KCl and control solutions (144–160 µm/s) than in those activated in CaCl₂ (104 µm/s); samples activated in NaCl yielded intermediate VCL values (127 µm/s). Post‐thaw sperm achieves maximum motility rate and velocities when activated in solutions composed of sodium citrate, NaCl, KCl or glucose. Thus, post‐thaw sperm motility of B. insignis can be triggered in ionic and non‐ionic solutions at osmolality between 0 and 200 mOsm/kg. The use of solutions containing calcium, however, should be avoided.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

Activation of sperm motility in striped bass via a cAMP-independent pathway

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.     

Unusual motility characteristics of sperm of the spotted wolffish

Unlike the sperm of most teleosts, that of the spotted wolffish Anarhichas minor is motile on stripping, remains motile for at least 2 days and loses motility when exposed to sea water. Computer assisted sperm analysis (CASA) was used to quantitatively examine the motility characteristics of spotted wolffish sperm. Straight line velocity (VSL), beat cross frequency (BCF) and percentage motility were the most sensitive indicators of movement. Sperm trajectories were very different to those of other teleosts examined, showing large side-to-side movements of the sperm head and a more 'wiggly' behaviour which may be an adaptation to swimming in the viscous gelatinous egg mass. VSL was not altered by pH from 5·0 to 9·0, but was lower at pH 4·5. It was highest at 200 to 500 mOsm and decreased rapidly at <200 mOsm and more slowly at >500 mOsm. It is suggested that the unusual characteristics of spotted wolffish sperm in its trajectory and duration of motility, its release in a fully activated state and its greatly decreased motility in both fresh and sea water are related to a spawning strategy involving mixing of sperm with eggs contained in a gelatinous mass rather than release directly into water in proximity to the ova.     

Purification, characterization and antigenic species-specific reactivity of vitellogenin of rosy barb (Puntius conchonius Hamilton)

Vitellogenin (Vg) was isolated using gel filtration and ion-exchange chromatography from plasma of rosy barb (Puntius conchonius) treated with estrogen (estradiol-17beta). The purified Vg was stained positive for carbohydrate, lipid and phosphorus and was rich in Ala (10.58%), Asp (8.46%), Glu (10.30%), Leu (11.23%), Lys (7.22%) and Val (7.49%). It appeared as a single band of approximately 450 kDa in native PAGE and was reduced to a single band of approximately 167 kDa under SDS-PAGE, suggesting that it is probably composed of three identical polypeptide subunits. Double-immunodiffusion assay showed that the plasma from female rosy barb reacted with the mouse antisera against rosy barb Vg, forming a single immunoprecipitin line, while the plasma from male rosy barb or female zebrafish showed no such reactivity, confirming the existence of the sex- and species-specific reactivity for rosy barb Vg antisera.     

Toxicity studies of nonylphenol on rosy barb (Puntius conchonious): a biochemical and histopathological evaluation

Acute and subacute toxicity of the nonylphenol (NP) on fish was investigated in laboratory toxicity tests with rosy barb (Puntius conchonious). The acute toxicity of NP to rosy barb was determined in semi-static bioassays. Median lethal concentration (LC50) for 96 h was 1.72 microM. The effects of sublethal concentrations of NP (0.17, 0.34 and 0.68 microM) on the structures and biochemical parameters [alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanin aminotransferase (ALT)] in gills, liver and kidney of rosy barb were studied after 14 days. The results showed that NP caused alteration of the structure in organs, as evidenced by the hyperplasia of epithelium and the fusion of secondary lamellae in the gills, the disappearance of the cell membrane and the cell necrosis in the liver as well as haemorrhages in the kidney. In addition, the functional enzyme activities were also changed. The increase trend in ALP activity in organs of fish treated with NP was recorded. The levels of AST and ALT in gills, liver and kidney were stimulated to rise at the lower concentration and fall at the higher concentration NP treatment compared to controls. This study suggests that NP can alter of the structures and biochemical parameters within non-endocrine tissue of fish and these changes may be mediated via destroying membrane structure and inducing cell necrosis.     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.